Obesity has been declared a first degree public health hazard since it is accompanied by hypertension, diabetes and cardiovascular diseases. Obesity is always accompanied by hypertrophy and/or hyperplasia of adipocytes. There are evidence to suggest that hormones and growth factors control the determination and differentiation of pre-adipose cells and that any qualitative and quantitative changes of these factors will create abnormal conditions leading to pathological states such as obesity. At the present time, very little is known about positive regulators of differentiation. The existence of yet unidentified factors in serum or in serum fraction playing an important role in the adipose differentiation has been repeatedly implied by many laboratories, including ours. The characterization of the adipogenic factor has not yet been carried out because of its difficulty. However, the investigation of its nature is of obvious importance since it has been shown that serum from obese patients has a higher ability to stimulate adipocyte differentiation than serum from normal subjects. Recently, we have found that normal hepatocytes in primary culture and the human hepatoma cell line HepG2 synthesize and secrete adipogenic activity in their culture medium. Comparison of the biochemical properties of adipogenic factor purified from serum and from HepG2-CM indicate that they are similar. We have also shown for the first time that adipogenic activity in liver extract was higher in obese mice than in their normal littermates. Since HepG2 cells can be grown in serum- free and factor-free medium, HepG2 conditioned medium (HepG2CM) is an ideal starting material for the purification of the adipogenic factor called L- ADF. L-ADF was purified 146,800 fold by a 4 step purification procedure with a recovery of 26%. We have obtained amino-acid sequence information corresponding to N-terminal 12 amino-acids of L-ADF. Search of protein database indicates that L-ADF is not homologous to any sequence in the database suggesting that it is a novel protein. These newly obtained data indicate that it is very important to pursue the molecular and biological characterization of L-ADF. Specifically, it is proposed to pursue structural study of L-ADF, obtain additional amino-acid sequence information in order to clone the L-ADF cDNA and develop anti-L-ADF antibody. Both molecular probes will also be used to investigate the level of L-ADF expression in normal and pathophysiological states of adipose tissue development in animal models and in human subjects. These studies will further our understanding of the regulation of adipose differentiation and provide tools towards the development of treatment of human obesity.